How to adorn trees with more cones: a lesson in patience Patrick von Aderkas & Lisheng Kong University of Victoria
Initial screening of hormone levels in apical buds where cone buds are formed Initially, 9 genotypes of coastal Douglas-fir 8 genotypes of lodgepole pine were tested. Over the years, we have measured hormones in at least double this number of genotypes of each species
♂ cone bud Year 1 Meiosis Year 2 Fertilization Microspores Pollination (pollen enters the ovule)
♀ cone bud Seed Year 1 Meiosis Fertilization Year 2 Megagametophyte only 1 of 4 megaspores survives Pollination (pollen enters the ovule)
Douglas-fir
♂ cone bud Year 1 Meiosis Year 2 Fertilization Microspores Year 3 Pollination (pollen enters the ovule)
♀ cone bud Seed Year 1 Meiosis Fertilization Year 2 Megagametophyte only 1 of 4 megaspores Year 3 survives Pollination (pollen enters the ovule)
• A photo of brach with three year cones Year 2 Year 3 Year 1
Do endogenous hormone concentrations change over the course of apical bud flush and the period of male and female cone initiation?
Resulted in very large studies with lots of repeated sampling, and lots of chemical analysis
Rather than take you through dozens and dozens of graphs, we will summarize the overall results. For the curious we have listed the papers in which they appear.
Publications (BCSOA folks in bold !) Kong, L., P. von Aderkas, Zaharia, L.I. 2016. Effects of exogenously applied gibberellins and thiadiazuron on phytohormone profiles of long-shoot buds and cone gender determination in lodgepole pine. Journal of Plant Growth Regulation 35: 172-182 Kong, L., P. von Aderkas, I. Zaharia, S.R. Abrams, T. Lee and J. Woods . 2012. Analysis of phytohormone profiles during male and female cone initiation and early differentiation in long-shoot buds of lodgepole pine. Journal of Plant Growth Regulation 31: 478-489. DOI 10.1007/s00344-011-9257-1 Kong, L., P. von Aderkas, S.J. Owen, B. Jaquish, J. Woods and S.R. Abrams. 2012. Effects of stem girdling on cone yield and endogenous phytohormones and metabolites in developing long shoots of Douglas- fir ( Pseudotsuga menziesii ). New Forests 43: 491-503. DOI 10.1007/s11056-011-9294-4 Kong, L., P von Aderkas, SJ Owen, T Wagner and SA Abrams. 2011. Comparison of endogenous cytokinins, ABA and metabolites during female cone bud differentiation in low and high cone-producing genotypes of lodgepole pine. Trees - Structure and Function 25: 1103-1110. DOI 10.1007/ s00468-011- 0585-3 Kong, L., S.R. Abrams, S. J. Owen, A. van Niejenhuis , P. von Aderkas. 2009. Dynamic changes in concentrations of auxin, cytokinin, ABA and selected metabolites in multiple genotypes of Douglas-fir ( Pseudotsuga menziesii ) during a growing season. Tree Physiology 29: 183-190. Kong, L., S.R. Abrams, S. J. Owen, H. Graham , P. von Aderkas. 2008. Phytohormones and their metabolites during long shoot development in Douglas-fir following cone induction by gibberellin injection. Tree Physiology 28: 1357-1364.
ABA catabolism
ABA catabolic changes in growing leader buds: Douglas-fir
ABA catabolic changes in growing leader buds: lodgepole pine
ABA catabolic pathways lodgepole pine & Doug-fir Doug-fir only
Concentration (ng/g DW) of endogenous hormones in lodgepole pine leader buds Early Late Cytokinin t-Z L L dhZ L L 10 t-ZOG L 61 + 2 43 + 7 t-ZR dhZR 37 + 2 27 + 5 14 + 1 11 + 1 iPA 11.4 + 0.5 2iP 7 Auxin 28 + 3 42 + 1 IAA IAAasp L L IAAglu L L np - no peak. L - below limit of quantification/calibration
Concentration (ng/g DW) of endogenous hormones in Douglas-fir leader buds early late Cytokinin t-Z L L dhZ L L 11.3 + 0.3 27 + 1 t-ZOG 34 + 1 172 + 1 t-ZR dhZR L 9.5 + 0.5 56 + 1 31.7 + 0.8 iPA 2iP L L Auxin 216 + 15 110 + 18 IAA IAAasp L L np np IAAglu np - no peak. L - below limit of quantification/calibration
Concentration (ng/g DW) of endogenous hormones in lodgepole pine leader buds Early Late Gibberellin np np GA1 np np GA3 np np GA4 np np GA7 np - no peak. L - below limit of quantification/calibration
Concentration (ng/g DW) of endogenous hormones in Douglas fir leader buds Early Late Gibberellin np np GA1 np np GA3 22 + 3 np GA4 np GA7 L np - no peak. L - below limit of quantification/calibration
Do endogenous hormone concentrations change over the course of apical bud flush and the period of male and female cone initiation? YES, in a quite species-specific manner ! Doug-fir lodgepole pine Cytokinins up down Auxins down up Abscisic acid down up Gibberellins not detectable not detectable
Conclusion Cone induction methods need to be designed in accordance with the temporal hormone regimes of a species
Conclusion Cone induction methods need to be designed in accordance with the temporal hormone regimes of a species BUT WAIT A MINUTE ! What about girdling and GA induction? How do they affect initiation of cones?
Douglas-fir 1. Optimization of female cone production 2. Induction of cones in cone-poor genotypes
Douglas-fir 1. Optimization of female cone production 2. Induction of cones in cone-poor genotypes
Girdling (alone) of 14-year-old Douglas-fir trees and its effect on endogenous hormones from time of treatment to end of initiation period two genotypes gibberellins (GA 4 ,GA 7 ) initial rise, then decline abscisic acid no change auxins no change cytokinins no change
Treatment 9550 9137 ♂ cone ♀ cone ♂ cone ♀ cone Control 0 0 572 + 143 0 Girdling 8723 + 1690 10 + 9 8135 + 1356 6 + 5
We then looked at the classic hormone injection treatment for Douglas-fir, GA 4/7 injection
4 7 3 1 prior to injection, GAs are, if even detectable, in the ng/g DW range – an order of magnitude less
4 7 3 1 prior to endogenous injection, GAs gibberellins are, if even – GA 1 & GA 3 – detectable, in are produced the ng/g DW range – an order of magnitude less
ABA cytokinins control 4 mg GA 4/7 control 4 mg GA 4/7 /tree /tree 40 mg GA 4/7 400mg GA 4/7 40 mg GA 4/7 400mg GA 4/7 /tree /tree /tree /tree
control 4 mg GA 4/7 /tree 40 mg GA 4/7 /tree IAA - auxin 400mg GA 4/7 /tree
Do endogenous hormone concentrations change after GA 4/7 injection (compared to controls) ? Cytokinins no change Auxins up three weeks after treatment Abscisic acid no change Gibberellins GA3 & GA1 up (endogenous) GA4 & GA7 up (endogenous & exogenous are impossible to separate)
♂ cone ♀ cone GA 4/7 (mg/tree) 0 2994 + 999 124 + 46 4 4367 + 1269 154 + 58 40 4917 + 1143 236 + 32 400 8166 + 1616 256 + 20
If you were given a choice of which hormones to play around with to try and improve cone production, the answer is clearly GA and auxins Cytokinins no change Auxins up three weeks after treatment Abscisic acid no change Gibberellins GA3 & GA1 up (endogenous) GA4 & GA7 up (endogenous & exogenous are impossible to separate)
Douglas-fir 1. Optimization of female cone production 2. Induction of cones in cone-poor genotypes
After years of trial and error.....
After years of trial and error, we came up with a winning combination for female cone induction – GA 4/7 with an anti-auxin, 2,3,5-triiodobenzoic acid (TIBA), which we tested on six historically poor-yielding i.e. crap, genotypes of Douglas-fir
Female cone yield per ramet in six Douglas-fir genotypes with various cone induction treatments. Mean ± SE, n= 3 to 5 for each genotype/treatment. Treatments were applied to six low yield genotypes by stem-injection. Treatment application was completed in spring 2013 in PRT seed orchard. Data was collected in spring 2014. GA- Gibberellin, MGBG- methylglyoxal bis (guanylhydrazone), TIBA- 2,3,5-triiodobenzoic acid, Put - putrescine
Female cone yield per ramet in six Douglas-fir genotypes with various cone induction treatments. Mean ± SE, n= 3 to 5 for each genotype/treatment. Treatments were applied to six low yield genotypes by stem-injection. Treatment application was completed in spring 2013 in PRT seed orchard. Data was collected in spring 2014. GA- Gibberellin, MGBG- methylglyoxal bis (guanylhydrazone), TIBA- 2,3,5-triiodobenzoic acid, Put - putrescine
* * five out of six poor- yielding genotypes did better ! * * * * * Female cone yield per ramet in six Douglas-fir genotypes with various cone induction treatments. Mean ± SE, n= 3 to 5 for each genotype/treatment. Treatments were applied to six low yield genotypes by stem-injection. Treatment application was completed in spring 2013 in PRT seed orchard. Data was collected in spring 2014. GA- Gibberellin, MGBG- methylglyoxal bis (guanylhydrazone), TIBA- 2,3,5-triiodobenzoic acid, Put - putrescine
Lodgepole pine 1. Hormone profiles 2. Optimization of female cone production
Lodgepole pine 1. Hormone profiles 2. Optimization of female cone production
A high-yielding genotype has a different hormone profile than a low-yielding genotype Hormone 233 (low) 299 (high) ABA high low cytokinins low high
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